1. Field of the Invention
The present invention relates to the fields of infectious disease, vaccines, molecular biology, and the treatment and prevention of cholera.
2. Description of Related Art
Vibrio cholerae is a Gram-negative bacterium that is responsible for the deadly diarrheal disease cholera. The bacterium expresses virulence factors within the human intestine that lead to intestinal colonization and disease symptoms. Two of the most important virulence factors are cholera toxin (CT), an ADP-ribosylating toxin that is largely responsible for the symptoms of disease, and the toxin coregulated pilus (TCP), a type IV pilus essential for intestinal colonization (Gill, 1976; Mekalanos et al., 1983; Pearson and Mekalanos, 1982; Herrington et al., 1988).
ToxT, an AraC family protein, activates the transcription of the genes encoding CT (ctx) and TCP (tcp), as well as the genes encoding other poorly understood “accessory colonization factors” (acf) (DiRita et al., 1991). Since toxT lies within the tcp gene cluster, ToxT is also able to regulate its own expression, allowing for continuous expression of ToxT under favorable conditions (Brown and Taylor, 1995; Yu and DiRita, 1999). V. cholerae strains lacking toxT express no CT or TCP, and fail to colonize the intestine (DiRita et al., 1996; Champion et al., 1997). ToxT binds to specific sites upstream of the ctxA, tcpA, acfa, tagA and aldA promoters (Eithey and DiRita, 2005; Withey and DiRita, 2005; Hulbert and Taylor, 2002) to stimulate transcription. The location of all of these binding sites is at least 45 bp upstream of the transcription startsite, with the exception of the ctxA promoter, where ToxT protects a region up to −13. This suggests that the tcpA, acfA, tagA, and aldA promoters are “Class I” AraC-like promoters (i.e., activated from sites that do not overlap with the −35 element), and consistent with this mode of activation, it has been demonstrated that ToxT-dependent tcpA transcription requires the C-terminal domain of the α subunit of RNAP (Hulbert and Taylor, 2002). However, the overlap of ToxT binding with the −35 element (albeit at lower affinity) at the ctxA promoter may indicate that this represents activation of a “Class II” AraC-like promoter, which has been shown in some AraC-like activators [e.g., RhaS, RhaR, MelR] (Wickstrum and Egan, 2004; Grainger et al., 2004) to involve contacts between the activator and σ70.
Recently, a “toxbox” motif (yrTTTTwwTwAww) has been identified within the ToxT-bound region at ToxT-activated promoters (Withey and DiRita, 2006). At all ToxT-activated promoters with the exception of aldA, two toxbox sequences are found in either a direct or inverted orientation. It has been shown that the insertion of 5 bp and 10 bp between the two toxboxes found at the acfa and tcpA promoters prevents transcriptional activation, but still allows ToxT binding in vitro (Withey and DiRita, 2005; Withey and DiRita, 2006). These results suggest that single ToxT monomers bind each toxbox, and that interactions between monomers are likely important for transcriptional activation.
AraC family members are classified based on homology to a 99 amino acid stretch within the AraC carboxy-terminus (Gallegos et al., 1997). The crystal structures of two AraC family proteins, MarA and Rob, complexed with DNA, have been resolved and the structures demonstrated that this region encodes two distinct helix-turn-helix (HTH) motifs that function in DNA-binding (Gallegos et al., 1997; Rhee et al., 1998; Kwon et al., 2000). MarA, Rob, and another protein, SoxS, are all able to bind to the same promoter elements (alternatively referred to as marbox/robbox/soxbox) and activate an overlapping set of genes, albeit with different affinities (Jair et al., 1996; Jair et al., 1995; Greenberg et al., 1990). The crystal structure of MarA bound to the mar promoter identified base- and phosphate backbone-specific contacts made between the first HTH (HTH1) and the 4 bp recognition element (RE)1 motif of the marbox, and specific contacts made between HTH2 and the 4 bp RE2 motif; RE1 and RE2 are centered on the same face of the DNA helix and separated by a 7 bp A/T-rich spacer. However, alanine substitution mutagenesis of MarA has suggested that while both HTH motifs contribute to DNA binding, the individual contributions of contacts made between HTH1 and RE1 are more important to DNA binding than HTH2-RE2 contacts (Gillette et al., 2000), and similar conclusions were drawn from alanine substitution mutagenesis of SoxS (Griffith and Wolf, 2002). Moreover, the crystal structure of Rob bound to the micF promoter indicated base- and phosphate backbone-specific contacts between HTH1 and RE1, similar to those found in the MarA-mar structure, but no base-specific contacts between HTH2 and RE2 and only one specific contact to the phosphate backbone (Kwon et al., 2000). It has been shown that bile acids interact with Rob to induce transcription (Rosenberg et al., 2003), so perhaps the apparent lack of specific contacts between HTH2 and RE2 may be due to a lack of inducer during crystallization. Studies of DNA binding by several AraC family members including AraC, MelR, RhaS, SoxS, and MarA have indicated that the orientation of the promoter-proximally bound activator depends on the distance to the −35 element, with the HTH2 oriented closest to the −35 element at Class II-like promoters (where the activator binding site extends downstream to at least −40) and with HTH1 oriented closest to the −35 element when the binding site is further upstream of the −35 element (Grainger et al., 2004; Porter and Dorman, 2002; Grainger et al., 2003; Behnde and Egan, 1999; Niland et al., 1996; Martin and Rosner, 2001; Martin et al., 1999; Wood et al., 1999).
The amino-terminus of AraC is responsible for dimerization and binding of the effector arabinose (Bustos and Schleif, 1993). The crystal structure of the amino terminus of AraC has also been resolved and revealed arabinose bound within an eight-stranded anti-parallel beta barrel “jelly roll” structure at the N-terminal end, and an antiparallel coiled-coil that mediates dimerization at the C-terminal end of this domain (Soisson et al., 1997). In general the N-termini of AraC family proteins (if present) do not share significant sequence homology, but they often share similar functions with the AraC N-terminus, such as oligomerization and/or effector binding (Gallegos et al., 1997; Martin and Rosner, 2001).
ToxT, like AraC, appears to contain two distinct domains: an N-terminal domain involved in dimerization and possibly environmental sensing, and a C-terminal domain necessary for DNA binding (Prouty et al., 2005). The ToxT N-terminus fused to the DNA binding domain of LexA is able to repress sulA transcription, consistent with dimerization determinants being located within the N-terminal domain. The ToxT N-terminus shares little sequence homology with the AraC N-terminus, but it is predicted to share secondary and tertiary structural similarities, as determined by Threading programs (Soding et al., 2005) (discussed below). Two different mutant forms of the N-terminus were identified that exhibited altered responses to the repressive effects of bile, suggesting that there are environmentally responsive elements within the N-terminus. The ToxT C-terminus is able to bind DNA, but only when fused to a heterologous dimerization domain, demonstrating that the C-terminus is sufficient for DNA binding, and that the C-terminus requires dimerization for DNA binding. Interestingly, the dimerized C-terminus is unable to activate transcription, suggesting that, unlike AraC and several other family members (Bustos and Schleif, 1993; Poore et al., 2001 there may be additional determinants in the ToxT N-terminus required for transcriptional activation.
This invention concerns the identification of amino acids critical for ToxT function. Specific amino acids involved in dimerization, DNA binding, and environmental modulation of ToxT have been identified. This information can be applied in the prevention and treatment of cholera.